Synthesis of Health-Promoting Carbohydrates
Verkhnyatskaya, Stella
DOI:
10.33612/diss.158661500
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Verkhnyatskaya, S. (2021). Synthesis of Health-Promoting Carbohydrates. University of Groningen. https://doi.org/10.33612/diss.158661500
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A straightforward glycosylation method is described to regio- and stereoselectively introduce two α-L-fucose moieties directly to the secondary rim of β-cyclodextrin. Using NMR and MS fragmentation studies, the nonasaccharide structure was determined, which was also visualized using molecular dynamics simulations. The glycosylation method proved to be robust on gram-scale and may be generally applied to directly glycosylate -cyclodextrins to make well-defined multivalent glycoclusters.
Chapter 3
Direct and Regioselective Di-α-Fucosylation on the
Secondary Rim of β-Cyclodextrin
Published in: Verkhnyatskaya, S. A., de Vries, A. H., Douma-de Vries, E., Sneep, E. J. L., Walvoort, M. T. C., Chem. Eur. J. 2019, 25, 6722.
34
3.1
Introduction
Carbohydrate-protein and carbohydrate-carbohydrate interactions are central to many biological processes in all domains of life, including glycoprotein folding, cell signaling, immunomodulation, and host-pathogen binding.1 Generally, these
interactions are highly multivalent in character, and distinct carbohydrate moieties spaced at specific distances are essential for productive binding.2 This ‘multivalency
effect’ has inspired the design of glycoconjugate clusters based on polymers,3
nanoparticles,4 cyclodextrins5,6 and calixarenes,7 amongst others, to which a plethora
of different glycans are attached.8 Interestingly, amongst the various attachment
methods and linkers, the natural glycosidic linkage is virtually absent. Instead, glycans are often attached through a peptide, thioester, or triazole moiety.
In an attempt to generate purely carbohydrate-based inhibitors of host-pathogen interactions for clinical and food applications, it was attempted to glycosylate cyclodextrins (CDs) directly with L-fucose moieties. Cyclodextrins are cyclic
oligosaccharides consisting of six, seven, or eight α-(1→4)-D-glucoside moieties, to give the so-called α-, β-, and γ- CDs, respectively. Owing to their non-toxic nature, CDs are generally regarded as safe (GRAS) and have received widespread attention in food, agriculture, cosmetics, and pharmacy. As depicted in Scheme 1, CDs generally adopt the shape of a truncated cone, which has the C-6 hydroxyls on the top (primary rim) and the C-2 and C-3 hydroxyls on the bottom of the cone (secondary rim, Scheme 1).9 Most
examples of chemically synthesized glycoclusters display the glycans on the primary rim,10-17 and only a few examples are reported of direct glycosylation of the hydroxyls. 18-21 Alternatively, enzymatic transglycosylation strategies resulted in the formation of a
mixture of multiply glycosylated CDs, bearing -galactosyl units on the C-2 or C-6 position of the glucosides.22,23 When two or more glycoside units were introduced, low
regioselectivity was observed. Thus, the application of chemical modifications allows for better control over the substitution pattern of CD.
Scheme 1. β-CD cone representation and schematic depiction of the secondary rim α-fucosylation
Interestingly, due to the primary alcohols, the primary rim is considered conformationally more flexible, resulting in a reduced effective diameter. In contrast, the alcohols on the secondary rim are sterically more constrained and less flexible, positioning the substituents in a well-defined manner. For the generation of multivalent CD-based glycoclusters, it is highly desirable to have a method to directly glycosylate
35 the secondary rim. The main challenges associated with directly glycosylating cyclodextrins arise from the lack of discrimination between the chemically similar hydroxyls on both rims, and the complicated characterization of the resulting products. Illustrative of the first challenge are the seminal reports from Sollogoub and co-workers that deal with the discrimination between the primary hydroxyls to install different functionalities.24 Also, several methods exist to regioselectively introduce or remove
one25 or two26-32 protecting groups on the primary rim. To the best of our knowledge,
methods to regioselectively liberate hydroxyls on the secondary rim are scarce, with one example reported for permethylated CD.33 In this Chapter, the stereoselective
di-α-fucosylation of β-CD on two C-3 positions of the secondary rim is reported. L-Fucose is
selected as a multivalent CD decoration for its central role in biological glycans,34-36
where it is attached to blood group antigens, the complex N-glycan of glycoproteins, and human milk oligosaccharides, amongst others. Fucosylated glycans have shown to block the intestinal adhesion of pathogenic bacteria such as Salmonella enterica37 and
Escherichia coli,38 and this makes multivalent fucosylated scaffolds interesting targets
for novel antimicrobial therapies.
3.2
Results and Discussion
Our method makes use of a β-CD ring (compound 4, Scheme 2) that has the C-3 hydroxyl groups of each glucoside unit available. The synthesis of the acceptors started with the perbenzylation of commercially available α- and β-CD, providing the protected CDs 1 and 2 in high yield (Scheme 2).30 These were then subjected to Et3SiH/I239,40 to
regioselectively liberate all C-3 hydroxyls to give acceptors 3 and 4, having six and seven free hydroxyls respectively.
Scheme 2. Synthesis of cyclodextrin acceptors 3 and 4
For the α-fucosylation of these acceptors, reported thio-fucoside donor 5 was utilized (Scheme 3). While the stereoselective introduction of multiple α-fucosidic bonds simultaneously remains a challenging endeavor, this donor has proven to provide robust α-fucosylation in other glycan syntheses.41 The first glycosylation
experiment was performed using 1.15 equivalents of donor 5 per hydroxyl (total: 6.9 eq for α-CD, 8.05 eq for β-CD), which was treated with pre-activation conditions (Ph2SO/Tf2O) at -80 °C (Scheme 3).42 After complete donor activation, the CD acceptor
was added and the reaction was left to proceed at -80 °C overnight, after which time the products were analyzed by UPLC-UV/MS (C4 column). To our surprise, the mixture of
36
products from the α-CD acceptor 3 contained more than 55% of the twice fucosylated octasaccharide, albeit a mixture of three isomers (peak f=2, Figure 1A, and Scheme 4A). The coupling constants of the anomeric fucose H1 signals indicated that the fucosidic linkage was formed stereoselectively, eliminating the possibility that the mixture of octasaccharides resulted from erosion of stereoselectivity. In addition, a significant amount of the (1→1’)-fucosyl disaccharide was isolated, probably resulting from donor hydrolysis and subsequent glycosylation, highlighting the poor reactivity of the CD acceptor. Interestingly, similar experimental conditions with the β-CD acceptor 4 also gave the twice fucosylated nonasaccharide 9 as the major product (50%), but it appeared as a single peak in the UPLC chromatogram, suggesting the formation of one major nonasaccharide isomer (Figure 1B). The α-stereoselectivity of the newly formed glycosidic bonds was confirmed by 1H NMR (J1-2= 4.0 Hz). To simplify the
characterization of the nonasaccharide product, compound 9 was isolated by preparative HPLC (20% isolated yield), and all benzyl protecting groups were removed by hydrogenolysis using Pd/C in THF/H2O to give product 10 (80%, Scheme 3).
Scheme 3. Fucosylation of CD acceptors 3 and 4, and synthesis of Fuc2-β-CD nonasaccharide 10 (numbering of Glc residues is given in blue)
37 Figure 1. UPLC-UV traces of fucosylated CDs after size-exclusion A) α-CDs B) Fuc-β-CDs
Scheme 4. Positional isomers of A) the protected octasaccharide (BnFuc2-α-CD) formed from the α-CD acceptor 3, B) unprotected isomers of nonasaccharide 10. The arrows depict the modified position in connection to residue A.
To elucidate the structure of the major nonasaccharide, three different regioisomers were considered (Scheme 4B): fucosides on the 3-OH positions of neighboring glucoside units (termed 3A,3B), two Fuc-Glc motifs separated by one unmodified glucoside (3A,3C), and two Fuc-Glc motifs separated by two unmodified glucose residues (3A,3D).1 The characterization efforts were commenced with NMR on
the purified sample. Signals arising from H-1, H-2, and H-3 within the ring of several Glc
1 The number in the name of one of the isomers refers to the modified position, in this case
38
and Fuc units were identified with 1H NMR, COSY, and TOCSY experiments for each
residue (see section 3.4.1). HSQC in combination with HMBC experiments provided the clear identification of the C-3 signals of the two Glc units to which a fucoside was attached. The latter signals shifted significantly downfield in HSQC as compared to the non-modified Glc-C3 signals (from 73.2-72.6 ppm to 77.4 and 77.2 ppm) and had a cross-peak with H-1 of fucoses in HMBC spectrum (Figure 2B, cross-peak A).
Also using HMBC analysis, the C4 signals of the modified Glc residues (Figure 2, bond and cross-peak B) were identified. Interestingly, these C4 signals had a cross-peak to H1 of a non-modified glucose residue (Figure 2A, bond C) indicating the presence of a non-fucosylated Glc residue linked to each of the Fuc-α(1→3)-Glc motifs, eliminating the possibility of the 3A,3B regioisomer (see section 3.4.1). However, further proof to determine the relative positions of the other unmodified Glc residues remained difficult to obtain, since many Glc signals overlapped. Also comparing it to the singly fucosylated β-CD, isolated from HPLC (BnFuc1-βCD, see Experimental Section), did not aid in the
characterization.
Figure 2. A) Fragment of the structure of compound 10with the identified cross-peaks H1Fuc → C3Glc (A, green bond), H3Glc → C4Glc (B, red bond), and C4Glc → 2xH1Glc’ (C, blue bond); B) Fragment of the HMBC NMR of 10.
Next, we turned our attention to mass spectrometry fragmentation studies.43
Ionization of fucosylated glycans in positive mode is notoriously prone to allow migration of the fucosides to neighboring residues upon MS analysis of intact protonated glycan ions, as recently highlighted by Seeberger and co-workers.44 In
contrast, the analysis of the deprotonated species formed with negative-mode ionization reduces the occurrence of side-reactions.45,46 Therefore, HILIC
chromatography in combination with MS in negative ion-mode was applied to fucosylated cyclodextrin 10, and parent masses of m/z 1425 [M-H]- and 712 [M-2H]
2-were observed. Next, fragmentation of the double charged ion was optimized, resulting in cross-ring fragmentations predominantly. In general, the CD ring is first opened to form linear parent ions, which will then undergo cross-ring fragmentations (Figure 3A).47,48 The observation of the consecutive 2,4A fragments 11, 13, 15, 17, 19,
and 21 confirms the presence of two glucoside units between the fucosylated residues, as in the 3A,3D type of modification (Figure 3B). Additionally, the m/z values
39 corresponding to the 0,2A and 0,2X fragments 23, 25, 26, 27, and 28 support the 3A,3D
substitution pattern by demonstrating evidence of three glucosides between the Glc-Fuc moieties (see section 3.4.2). Specific fragments corresponding to the 3A,3C substitution pattern were not observed.
Figure 3. A) Fragmentation pattern of two possible parent ions; B) Low-resolution MS/MS spectrum of nonasaccharide 10 (region from m/z 500 to 1150) measured on LCQ Fleet mass spectrometer (Thermo Fisher Scientific) in combination with an ion-trap
The clear regioselectivity of fucosylation on β-CD encouraged the optimization of the reaction conditions to increase the yield of the twice fucosylated product 9. The reaction times and temperatures were varied while keeping the equivalents of donor 5, acceptor 4, Tf2O, Ph2SO, and TTBP unchanged (Table 1). First, conditions were changed
towards a shorter reaction time and higher temperature: -70 °C for 3 hours (compared to -80 °C overnight). These conditions provided a mixture containing 33% of the di-fucosylated product. However, the formation of higher di-fucosylated compounds, i.e. tetra- and pentafucosylation, had significantly increased. Next, the reaction was started at -80 °C followed by warming the mixture to different temperatures (-70 °C, -60 °C and -50 °C), resulting in short reaction times (10-30 minutes). Quenching the reaction at -70 °C resulted in 35% of the nonasaccharide, while a significant amount of unreacted acceptor was still present (19%) together with mono-fucosylated Fuc1-β-CD product
(24%). Increasing the temperature to -60 °C provided 57% of the desired di-fucosylated product while quenching the reaction at -50 °C led to a reduced contribution of di-fucosylated product (35%). As a result, the highest yield of nonasaccharide 9 was observed when the reaction was neutralized at -60 °C, and these
40
conditions were used to α-fucosylate the -cyclodextrin acceptor on a large scale (1 g, 0.42 mmol, Table 1, entry 6).
Table 1. Optimization of the glycosylation conditions
Entry Reaction Conditions Bnβ-CD (4) BnFuc1 -β-CD BnFuc2 -β-CD (9) BnFuc3 -β-CD BnFuc4 -β-CD BnFuc5 -β-CD 1 -80 °C (o/n) 2 5 46 27 15 5 2 -70 °C (3h) 5 9 33 21 20 12 3 quenched at -70 °C 19 24 35 15 7 nd 4 quenched at -60° C 5 12 57 18 9 nd 5 quenched at -50 °C 2 5 35 29 20 9 61 quenched at -60 °С 6 13 61 20 6 nd
1 Performed on large scale
These optimized conditions provided a mixture of fucosylated CDs in 95% yield containing 61% of nonasaccharide, which could be purified by preparative HPLC (purity >90%), or by regular flash column chromatography, providing nonasaccharide 9 in 67% isolated yield and in 69% purity. After global deprotection, 275 mg of compound 10 was obtained (75%).
Structural information of the fucoside positioning in space is important to rationalize future carbohydrate-lectin binding so in an effort to understand the spatial arrangement of di-fucosylated β-CD 10, molecular dynamics (MD) simulations were performed. Since a recent comprehensive study of native CDs in water showed that structural and dynamic properties may differ depending on the choice of force field, two different force fields,49 i.e. GROMOS 53A650 and Q4MD,51 were used in combination with
the GROMACS simulation package.52 GROMOS is a so-called united atom model, in which
aliphatic H-atoms are not treated explicitly, but modeled effectively together with the C-atom they are attached to,50 while Q4MD is an AMBER-based all-atom force field,
optimized for carbohydrates, with a version specific for cyclodextrins (Q4MD-CD) which is used here.51 In contrast to what is commonly assumed, the cyclodextrin cone
is quite flexible, especially in protic solvents.53
This is apparent from the tumbling of glucoside units, which bend outwards and distort the conical shape and the hydrogen-bonding network. Indeed, our analysis reveals that specifically the fucosylated glucoside units show enhanced rotation and increased chair flexibility. These tilted states are not frequent but are stable over several nanoseconds (Figure 4B). In both force fields, the shape of the barrel is disturbed, i.e. glucopyranose residues tilt out of the barrel-shape; these tilts are mostly seen as concerted changes in two neighboring dihedral angles connecting consecutive sugars (one dihedral angle becomes positive, while the other becomes negative, shown in boxes on Figure 4B). Interestingly, the dynamics of the two force fields differ in this tilting, with the GROMOS force field being the less dynamic one. The study of Gebhardt et al. for a native (i.e. unsubstituted) β-CD shows that force fields differ in this aspect
41 both concerning thermodynamics and kinetics, but generally do show (partial) tilting of the sugars with respect to the barrel wall.49
Figure 4. (A) The O5-C1-C4-C5 dihedral angles connecting the ring Glc residues ; (B) Dihedral angles between two neighboring glucose residues within the β-CD rings of nonasaccharide 10 as a function of time for the combined trajectory. 0-4000 ns: GROMOS force field; 4000-8000 ns: Q4MD force field. The dashed vertical bar at 4000 ns separates the runs with the two force fields. Coloring is based on the naming of the β-CD ring sugars. For example, blue represents the dihedral between rings G and A (for the naming of the rings, see Scheme 3), with the dihedral angle defined by the O5-C1-C4-C5 atoms as shown in Figure 4.
The tilting of the cyclodextrin ring sugars is more pronounced upon fucosylation (Figure 5A and 5B), which can be seen from the enhanced probability of finding the dihedral angles connecting Glc units G-A (blue dashed line) and C-D (orange dashed line) at values of 60-90 degrees away from the main peak. Both force fields show this effect, but the Q4MD force field shows it more strongly, which is a reflection of the larger conformational flexibility found using this force field. This flexibility is also apparent from the wider distributions of the dihedral angles (Figure 5B). The conformations visited during the simulations were grouped into distinguishable conformational states (so-called clusters) by clusteranalysis.2
2 For more details on the modelling please refer to the original paper: S. A. Verkhnyatskaya,
42
Figure 5. Comparison of the distribution of dihedral angles between two neighboring Glc residues within the β-CD rings between β-CD and nonasaccharide 10. Dihedral angle distributions for natural β-CD are depicted with solid lines and fucosylated β-CD 10 with dashed lines, using both the GROMOS force field (A) and the Q4MD force field (B) The cluster analysis (Figure 6) shows that there is clearly a most predominant conformational state, making up 67% of all conformations in the combined trajectories (Figure 6, top left), which is a canonical β-CD barrel with the two fucose moieties slightly tilted inward from the barrel wall, but not residing within the barrel. Within GROMOS, this is the overwhelmingly dominant conformational state, with 93% abundance. In the Q4MD force field it is also clearly the most abundant state, but contains only 41% of all conformations.
The most predominant non-canonical structures are the ones where the fucosylated residues (GlcA and GlcD) tilt out of the barrel by about 90 degrees: in the
second most populated cluster (Figure 6, cluster 2) one of the fucosylated sugars is tilted outward, while in the third most populated cluster (Figure 6, cluster 3) this hold for both fucosylated sugars. The contribution of these conformations from the GROMOS force field is very low. In contrast, the sixth cluster is predominantly found in the GROMOS run and hardly in the Q4MD run. Thus, in the most abundant conformations the fucosides are attached to a canonical CD ring and prefer to be slightly tilted inward or outward with respect to the CD cavity. The distance between the two centers of the fucosides is peaked at ~7 Å, but the groups can occasionally be considerably closer at ~5 Å, or more distant depending on the tumbling of sugar rings and rotations of the fucoses with respect to the β-CD barrel wall.
43 Figure 6. Representative snapshots of major conformational clusters. Representative snapshots viewed from the bottom (left) and side (right) from the first six conformational clusters obtained from 16,000 conformations from the combined trajectories. The numbers indicate the rank of the cluster (bold yellow), the percentage of conformations in this cluster contributed to the cluster from the combined (yellow), GROMOS (orange), and Q4MD (magenta) trajectories, respectively.
In an effort to confirm the structural representation from MD simulations with NMR analysis, NOE intensities were calculated. The MD simulations suggested the presence of weak NOE signals that reflect the conformational flexibility of nonasaccharide 10, in particular regarding the different orientations of the fucosides. The H-atoms on the fucose C5 and C6 positions (H5FucA,D and H6FucA,D) are relatively
close to the H-atom on C3 of the upstream glucoside (H3GG,C) in many of the most
populated clusters (section 3.4.3). The closest distance between H5FucA,D and H3GG,C is
~1.9 Å in the most populated cluster, and the closest distance between the H6FucA,D and
44
the representative structure of the most populated conformational cluster in Figure 7A. Excitingly, these NOE cross-peaks were indeed observed experimentally in the NOESY spectrum as shown in Figure 7B, supporting the dynamical ensemble observed in the MD simulations. The MD analysis also suggests that the bending of the Fuc rings away from the CD barrel is associated with enhanced chair flexibility of the substituted Glc rings (residues A and D). The relatively low 3J couplings of 8.3 Hz measured for these
Glc H3 hydrogens in compound 10, as compared to 9.5 Hz for the H-3 signals in natural β-CD, are consistent with the MD simulation, and support the contribution of the unfavored 1C4 conformer.54 The MD analysis shown here should be taken as qualitative:
the dynamics of the conformational changes are slow on the MD time-scale, and it is not possible to extract converged thermodynamic (equilibrium) data from the extensive simulations of 4 microseconds.
Figure 7. A) Characteristic conformation of 10 with H-5Fuc, H-6Fuc and H-3Glc highlighted as black spheres for close contact between fucose on Glc(A) and Glc(G) (fucoside C-atoms are shown in dark blue); B) Observed NOE of H-5Fuc(A,D) → H-3Glc (G,C)
3.3
Conclusions
This Chapter presents the regioselective 3A,3D-fucosylation of the secondary rim of semi-protected β-CD, which proved to be robust, reproducible, and scalable. In natural glycans, such as HMOs, the fucosides are often separated by non-fucosylated carbohydrate residues, making this 3A,3D pattern a highly relevant mimic. While the regioselective 6A,6D substitution of CDs is well-developed for decoration of the primary rim,30,55 only limited examples of di-substitutions on the secondary rim exist,56 and
none of these examples covers a direct glycosylation reaction. It is proposed that the regioselectivity observed here arises from the kinetic conditions employed in the glycosylation reaction, i.e. low temperatures and short reaction times. This may drive the reaction to yield the least sterically hindered 3A,3D nonasaccharide as the kinetic product of the reaction. It is hypothesized that the remarkable regioselectivity observed for the β-, but not for the α-CD, is likely to depend on the size of the CD ring. MD simulations to corroborate this hypothesis are covered in Chapter 4.
45
3.4
Acknowledgments
Dr. Alex de Vries is acknowledged for performing the MD simulations. Elmatine Douma-de Vries is acknowledged for the contribution to the synthesis of the building blocks. Renze Sneep is acknowledged for optimization of the LC-MS/MS experiments.
3.5
Supporting Information
3.5.1 A detailed explanation of the NMR assignment
Scheme S1.Fucosylated region of CD. NMR experiments used to identify certain linkages are presented in two colors: green – TOCSY, red – HMBC.
To identify the connections within a fucosylated glucoside ring, HH-COSY (Figure S1) was used: first, the H1Glc → H2Glc correlations were identified, but further H2Glc →
H3Glc correlations were overlapping. Thus, a TOCSY (Figure S2) experiment was used
to identify the H1Glc → H3Glc correlations, and two overlapping H-3 signals were
identified at 4.15 ppm, and a cross-peak resulting from H1Fuc → C3Glc was observed in
an HMBC experiment (Figure S3, linkage A), providing confirmation that the fucoside unit was attached through an α-linkage to the C-3 of the glucoside. These C3Glc signals
have a 13C signal at 77.5 ppm and an H-3 signal at 4.15 ppm (based on HSQC, Figure S4).
From HMBC experiments (Figure S3, linkage B) the connection from H3Glc → C4Glc was
found, with the corresponding C4Glc signals at 78.5 ppm. Using the HMBC experiment
(Figure S3, linkage C), the C4 signals appeared to have a cross-peak at 5.08 ppm, which connected them to H1Glc’ (a so-called upstream glucoside unit). The peak at 5.08 ppm
appears as an app. triplet with integral = 2, so it was identified as two H1Glc’ signals.
TOCSY correlations (Figure S2) revealed that these signals coupled to H3Glc’ signals that
were not linked to a fucoside, leading to the conclusion that each of the (Fuc)Glc motifs has a neighboring non-modified residue, eliminating the 3A,3B modification pattern.
46
Figure S1. COSY NMR (600 MHz, D2O), showing the region with H1Glc→ H2Glc and H1Fuc → H2Fuc correlations. Further on all figures in the section: H-peaks of fucoses are highlighted with green, fucosylated glucoses with red, and non-fucosylated with blue.
Figure S2. TOCSY (600 MHz, D2O) mixing time 80 ms. The H1Glc→ H3Glc and H1Fuc→ H3Fuc correlations are framed.
47 Figure S3. HMBC (600 MHz, D2O) the following connections are shown in frames: A) 2xH1Fuc → 2xC3Glc (green bonds) B) 2xH3Glc → C4Glc (red bonds) C) 2xC4Glc → 2xH1Glc’ (blue bonds)
48
3.5.2 A detailed explanation of the MS/MS experiments
Upon fragmentation of the CD, an opening of the ring occurs, forming a parent-ion precursor (Scheme S2). The parent ion can undergo different types of cleavages leading to 2 types of adducts: cross-ring fragments (2,4A/2,4X and 0,2A/0,2X series) and fragments
originating from glycosidic bond cleavages (B, Y, C, and Z). In the MS/MS spectrum (Figure S5) three major fragments were identified (Scheme S3): two were originating from cleavage of one fucoside unit, leading to Z- and Y-ions (1262 and 639), and a cross-ring 2,4A-fragment resulting from cleavage of the reducing end glucoside (652).
For the modified β-CDs, seven different possibilities of opening the CD ring are possible, with many similar fragments originating from these options. For the 3A,3C isomer only the linear parent ion depicted in Scheme S4 leads to characteristic fragments (i.e. fragments not possible with the 3A,3D isomer), that have m/z values of 717, 707, 657, and 767 (Table S1). These fragments are not observed in the MS/MS spectrum (Figure S5). For the 3A,3D regioisomer three linear parent ions were considered (Scheme S5) and for linear parent ion II several ions corresponding to glycosidic bond fragments were observed (Table S2, values found are highlighted in red). Fragments originating from cross-ring cleavages were dominating in the MS/MS spectrum and are given in Table S3, where all the m/z values that were found in MS/MS spectrum are shown in red. Because the pattern of found m/z values only confirm the presence of the 3A,3D isomer, and characteristic fragments for 3A,3C isomer were not found, it was concluded that nonasaccharide 10 is substituted in a 3A,3D fashion.
49 Scheme S2. Possible initial fragmentation to form the linear parent ion. Typical oligosaccharide fragmentations are shown with arrows.
50
Scheme S4. Linear parent ion leading to characteristic 3A,3C fragmentations
Parent molecule AC Ring 0,2A 0,2X 2,4A 2,4X A 248 1177 - - B 409 1015 349 1075 C 717 707 657 767 D 879 545 819 605 E 1041 383 981 443 F 1203 221 1143 281
Table S1. Cross-ring fragmentations for the 3A.3C nonasaccharide isomer. Characteristic m/z values are shown in blue.
51 Scheme S5-II. Parent II of the 3A,3D isomer
Scheme S5-III. Parent III of the 3A, 3D isomer
Table S 2. M/z values of the fragments originating from glycosidic bond cleavages. M/z values found in the MS/MS spectrum are shown in red
AD from parent II B Y C Z 145 1280 160 1264 452 972 468 956 614 810 630 794 776 648 792 632 1084 340 1100 324 1246 178 1262 162
52
Table S 3. M/z values of the fragments from different linear parent ions of the 3A,3D isomer. M/z values found in the MS/MS spectrum are shown in red.
I II III 0,2A 0,2X 2,4A 2,4X 0,2A 0,2X 2,4A 2,4X 0,2A 0,2X 2,4A 2,4X 409 1015 349 1075 409 1015 349 1075 264 1161 203 1221 571 853 511 913 571 853 511 913 571 853 511 913 733 691 673 751 733 691 673 751 733 691 673 751 1041 383 981 443 895 529 835 589 895 529 835 589 1203 221 1143 281 1203 281 1143 281 1057 367 997 427 1365 59 1305 119 1365 59 1305 119 1365 59 1305 119
3.5.3 Computational Details
Figure S6 shows the most prominent candidates for close contacts with the fucose on ring A (for definition, see Scheme 3). The H-atoms on the fucose C5 and C6 (H5,6FA) are relatively close to the H-atom on C3 of the upstream barrel sugar (H3G) in many of the most populated clusters. The closest distance between H5FA and H3G is 1.9 Å in cluster 1; the closest approach distances between the H6FA and H3G are about 2 Å. The most common conformational variation of the fucose is the turning with respect to the cyclodextrin barrel, cf. clusters 1 and 2-4 in Figure S6. In this conformational variation of the fucose, the H-atom on C1 of fucose (H1FA) approaches H3G; the closest distance is 2.28 Å in cluster 2.
Figure S6. Non-trivial NOE candidates for characterization of conformational diversity of β-Fuc2-CD(3A,3D) (compound 10), specifically looking at the fucose on ring A. The figure shows snapshots of the eight most populated clusters of the simulation in the Q4MD model, highlighting the atoms involved in the close approaches. Magenta: H3 on residue G, yellow: H5 and H6s on the fucose attached to residue A (downstream of G); orange: H1 on the fucose attached to residue A.
53 Similarly, Figure S7 shows the most prominent candidates for close contacts with the fucose on ring D. The H-atoms on the fucose C5 and C6 (H5,6FD) are relatively close to the H-atom on C3 of the upstream barrel sugar (H3C) in many of the most populated clusters. The closest distance between H5FA and H3G is 2.06 Å in cluster 4; the closest approach distances between the H6FD and H3C are about 2.5 Å. The most common conformational variation of the fucose is the turning with respect to the cyclodextrin barrel, cf. clusters 1 and 2 in Figure S8. In this conformational variation of the fucose, the H-atom on C1 of fucose (H1FD) approaches H3C; the closest distance is 2.44 Å in cluster 1. In addition, there is a close approach to H3C by the H-atom on C3 of the fucose (H3FD); the closest distance is 2.43 Å in cluster 5.
Figure S7. Non-trivial NOE candidates for characterization of conformational diversity of β-Fuc2-CD (3A,3D) (compound 10), specifically looking at the fucose on ring D. The figure shows snapshots of the eight most populated clusters of the simulation in the Q4MD model, highlighting the atoms involved in the close approaches. Magenta: H3 on residue C, yellow: H5 and H6s on the fucose attached to residue D (downstream of C); orange: H1 on the fucose attached to residue D; green: H3 on the fucose attached to residue D.
3.5.4 General Experimental Procedures
All solvents used were of commercial grade and used without further purification. Dry DCM, toluene, and THF were generated by an MBraun SPS 800 solvent purification system. Solvents used for workup and column chromatography were of technical or HPLC grade from Boom, Biosolve, or Honeywell and used as purchased. Solvents were removed by rotary evaporation under reduced pressure at 45°C. Reagents were purchased from Sigma-Aldrich, Acros, TCI Europe, or CarboSynth and used without further purification. Reaction temperature refers to the temperature of the cooling bath equipped with a stirring bar unless stated otherwise. Reactions were monitored by TLC analysis on Merck silica gel 60/Kieselguhr F254 and spots were visualized by UV light, or spraying with orcinol stain 180 mg orcinol, 10 mL 85% H3PO4, 5 mL
EtOH, and 85 mL H2O) or with Seebach’s stain (2.5 g phosphomolybdic acid, 1 g Ce(SO4)2, 6 mL
H2SO4 and 94 mL H2O) followed by heating with a heat gun. Column chromatography was
54
GmbH, Germany) as the stationary phase. Size-exclusion chromatography was performed on Sephadex LH-20 using DCM/MeOH (1/1, v/v) as eluent. Molecular sieves 4Å (Merck, Germany) were activated by heating with a heat gun in vacuo.
1H and 13C NMR spectra were recorded on a Varian 400-MR (400/100 MHz) or a Bruker
Avance NEO (600/150 MHz). Chemical shifts are given in ppm with the solvent resonance as internal standard (CDCl3: δ7.26 for 1H, δ 77 for 13C; D2O: δ 4.79 for 1H). All individual signals
were assigned using 2D NMR spectroscopy: HH-gCOSY, gHSQC or NOE. Data are reported as follows: chemical shifts (δ), multiplicity (s = singlet, d = doublet, dd = double doublet, ddd = double double doublet, t = triplet, q = quartet, p = quintet, m = multiplet, apparent quartet = app q), coupling constants J (Hz), and integration. High-resolution mass measurements were performed on an LTQ Orbitrap XL spectrometer (Thermo Fisher Scientific) with an ESI ionization source.
UPLC-UV/MS measurements were performed on a Vanquish UHPLC system coupled to an LCQ Fleet mass spectrometer (Thermo Fisher Scientific) using an Acquity UPLC BEH C4 column (Waters, 2.1×150 mm, 1.7 µm) in combination with eluents A (10 mM ammonia acetate in H2O)
and B (acetonitrile), or using a Cortecs UPLC HILIC column (Waters, 2.1×150 mm, 1.6 µm) in combination with eluents A (H2O + 0.025% NH4OH) and B (acetonitrile + 0.025% NH4OH). The
UV traces were measured at 208 and 254 nm simultaneously and the ionization was performed with an ESI-source. In positive mode, the [M+NH4]+ or [M+2NH4]2+ was most prominent. In
negative mode, the [M-H]- or [M-2H]2- was most prominent. For chromatography on the C4 a 25
min run (flow rate 0.3 mL/min) was used, and the gradient used in the method was as follows: from 70% to 95% of B (from 0 to 20 min) and 70% of B (from 21 to 25 min). For chromatography on the Cortects HILIC a 25 min run (flow rate 0.3 mL/min) was used, and the gradient was as follows: 70% of B (from 0 to 2 min), from 70% to 50% of B (from 2 to 15 min) and 70% of B (from 16 to 25 min).
For preparative HPLC purification (Shimadzu), an Xbridge BEH C4 column (Waters, 10×150 mm, 5 µm) was used in combination with eluents A (H2O) and B (acetonitrile) with UV-detection
at 208 nm. For purification, a sample was dissolved in acetonitrile in a concentration of 2 mg/mL, and up to 800 µL was injected without affecting the separation. A 15 min column flush with 70% of B was performed before each run. A method of 40 min (flow rate 5 mL/min) was used and the gradient was as follows: from 70% to 95% (from 0 to 30 min) and 95% of B (from 30 to 40 min). For fragmentation experiments, separation on Cortects HILIC was performed. Samples were prepared in 70% acetonitrile in water in a concentration of 1 mg/mL, and 10 µL was used per injection. Upon chromatography an eluent was introduced into ESI ion source where the following conditions were set: heater temperature 75 °C, spray voltage 2 kV, capillary temperature 200 °C, capillary voltage -2 V. Collision induced dissociation was set at 25% normalized collision energy and an isotopic width of 3 m/z. The MS/MS spectra were obtained in negative ion mode in a mass range from 195 to 1440 m/z.
3.5.4.1 Synthesis of fucoside donor 5
Scheme S5. Synthesis of fucoside donor 5. Reagents and conditions: i) 1. Ac2O, pyridine, 0°C to RT 2. 33% HBr/AcOH 3. HSTol, TBAB(aq), KOH (aq), CHCl3; ii) Na in MeOH; iii) BnBr, NaH, DMF, 0 °C to RT
55 4-Methylphenyl 2,3,4-tri-O-acetyl-1-thio-β-L-fucopyranoside (F1).
L-(–)-fucose (5.03 g, 31 mmol) was portionwise added to a stirred solution of pyridine (54 mL, 52.8 g, 0.668 mol) and acetic anhydride (42 mL, 45.4 g, 0.444 mol) under nitrogen at 0 °C over the course of 15 min. The reaction was left to stir overnight at +4 °C after which time it was poured on a mixture of crushed ice and water. The aqueous layer was extracted with DCM (3x). The combined organic layers were washed with H2O (2x), brine, dried over MgSO4 and concentrated in vacuo. The crude compound
was flushed through a pad of silica gel (pentane/EtOAc = 2/1) and concentrated in vacuo. Next, a mixture of peracetylated fucose (~31 mmol) in DCM (120 mL, 0.25 M) was cooled to 0 °C under nitrogen, and HBr (33 wt. % in acetic acid, 40 mL, 56.0 g, 0.692 mol) was added dropwise in 20 min. When TLC analysis indicated complete consumption of the starting material (2.5h) the reaction mixture was carefully poured on crushed ice and stirred. The resulting aqueous layer was extracted with DCM (2x). The combined organic layers were washed with NaHCO3 (3x) until
neutral pH, then washed with H2O (1x), brine (1x), dried over MgSO4,and concentrated in vacuo.
Without further purification the resulting anomeric bromide was dissolved in CHCl3 (300 mL)
and p-thiocresol (5.5 g, 44 mmol) and TBABaq (dissolved in 47 mL H2O, 1.88 g, 5.8 mmol) were
added. To the reaction mixture KOHaq (dissolved in 45 mL H2O, 3.4 g, 61 mmol) was added
dropwise in 10 min. The two-layered reaction mixture was stirred vigorously overnight at ambient temperature. TLC analysis (DCM/MeOH/H2O, 10/5/1, v/v/v) indicated complete
consumption of the anomeric bromide. The organic layer was washed with H2O (1x), brine (1x),
dried over MgSO4,and concentrated in vacuo. Purification by flash column chromatography
(silica gel, gradient from pentane/EtOAc, 6/1 to 2/1) afforded the title compound as a white solid (Yield: 10.62 g, 26.8 mmol, 89% over 3 steps). The analytical data were in accordance with those reported previously.57 TLC: Rf 0.21 (pentane/EtOAc, 6/1, v/v).
4-Methylphenyl 1-thio-β-L-fucopyranoside
(F2).
To a stirred solution of monosaccharide F1 (7.52 g, 19.0 mmol) in anhydrous MeOH (40 mL) a piece of sodium (approx. 300 mg) was added. When TLC analysis (DCM/MeOH, 5/1, v/v) indicated complete consumption of the starting material (25 min) the reaction mixture was neutralized by the addition of Amberlite IR-120H+ until neutral pH, filtered and concentrated in vacuo. The title compound was obtained as
a white solid (Yield: 5.01 g, 18.5 mmol, 98%). The analytical data were in accordance with those reported previously.58 TLC: Rf = 0.33 (EtOAc).
4-Methylphenyl 2,3,4-tri-O-benzyl-1-thio-β-L-fucopyranoside
(5).
To a stirred solution of p-methylphenyl 1-thio-β-L-fucopyranoside (8.72 g, 32 mmol) in DMF (108 mL) under nitrogen atmosphere, NaH (60% dispersion in mineral oil, 6.6 g, 0.165 mol) was added. The reaction was stirred for 20 minutes, after which time benzyl bromide (20 mL, 0.165 mol) was added dropwise in 1 h. The reaction mixture was stirred overnight at ambient temperature (18h) and then diluted by Et2O and quenched by the slow addition of an ice-water mixture. The aqueous layer was
extracted with Et2O (3x). The organic layer was washed with brine, dried over MgSO4, and
concentrated in vacuo. Recrystallization from EtOH afforded the title compound as a white fluffy solid (Yield: 13.8 g, 25 mmol, 79%). The analytical data were in accordance with those reported previously.59,60 Rf = 0.33 (pentane/Et2O, 6/1, v/v).
1H NMR (400 MHz, CDCl3) δ 7.50 – 7.47 (m, 2H, CHarom STol), 7.42 – 7.27 (m, 13H, CHarom Bn), 7.01
(d, 2H, J = 7.8 Hz, CHarom STol), 5.00 (d, 1H, J = 11.6 Hz, CHH Bn), 4.80 (d, 1H, J = 10.2 Hz, CHH
Bn), 4.75 – 4.70 (m, 3H, CHH Bn, CH2 Bn), 4.66 (d, 1H, J = 11.6 Hz, CHH Bn), 4.54 (d, 1H, J = 9.6
Hz, H-1), 3.89 (t, 1H, J = 9.4 Hz, H-2), 3.63 (d, 1H, J = 2.8 Hz, H-4), 3.58 (dd, 1H, J = 9.2, 2.8 Hz, H-3), 3.51 (q, 1H, J = 6.4 Hz, H-5), 2.30 (s, 3H, CH3 STol), 1.26 (d, 3H, J = 6.3 Hz, H-6).
56
13C NMR (100 MHz, CDCl3) δ 138.8, 138.5, 138.4, 137.1 (Cq), 132.2, 130.5, 129.5, 128.4, 128.3,
128.3, 128.1, 128.0, 127.7, 127.6, 127.4 (CHarom), 87.9 (C-1), 84.6 (C-3), 77.2 (C-2), 76.6 (C-4),
75.5 (CH2 Bn), 74.6 (C-5), 72.8 (CH2 Bn), 21.1 (CH3 STol), 17.3 (C-6).
3.5.4.2 Synthesis of the cyclodextrin acceptors
Scheme S6. Synthesis of CD acceptors. Reagents and conditions: i) for α-CD: BnBr, NaH, DMF; for β-CD: BnCl, NaH, DMSO; ii) I2, Et3SiH, DCM, -60 ° to -35 °C.
Perbenzylated α-CD (1).
To a stirred solution of α-cyclodextrin (1.05 g, 1.08 mmol) in DMF (13.5 mL) under nitrogen at 0 °C, NaH (60% dispersion in mineral oil, 4.7 g, 117.5 mmol) was added portionwise. After stirring the reaction mixture for 15 min, benzyl bromide (14 mL, 118 mmol) was added to the solution dropwise in 30 min. After 4.5h when TLC analysis indicated complete conversion of the starting material into a single spot, the reaction mixture was diluted with Et2O and
quenched by the slow addition of ice-water at 0 °C. The water layer was extracted with Et2O (3×), the combined organic layers were washed with H2O, brine, dried over
MgSO4,and concentrated. Purification by flash column chromatography (silica gel, pentane/Et2O,
6/1 to 2/1) afforded the title compound as a white foam (Yield: 2.43 g, 0.94 mmol, 87%). The analytical data were in accordance with those reported previously.30 Rf = 0.41 (pentane/Et2O,
2/1, v/v).
2A-F, 6A-F-dodeca-O-benzyl-α-CD (3).
Perbenzylated α-cyclodextrin 1 (580 mg, 0.23 mmol) and iodine (379 mg, 1.5 mmol) were suspended in DCM (83 mL, 18 mM I2) under nitrogen. After the
complete dissolution of iodine, the purple reaction mixture was cooled down to -45 °C (temperature measured inside the flask), followed by the addition of triethylsilane (0.24 mL, 1.5 mmol). The reaction mixture was allowed to warm up to -35 °C (1.5h) and was then neutralized by the addition of solid K2CO3 and
then the reaction mixture was washed with a saturated solution of Na2S2O3.
The water layer was extracted with DCM (2x) and the combined organic layers were washed with brine, dried over MgSO4, and concentrated. Purification using flash column chromatography
(silica gel, toluene/EtOAc, from 9/1 to 5/1, v/v) yielded the title compound as a white foam (Yield: 279 mg, 0.14 mmol, 60%). The analytical data were in accordance with those reported previously.40 TLC: Rf = 0.44 (toluene/EtOAc, 5/1, v/v).
β-CD
1H NMR (400 MHz, D2O) δ 5.03 (d, J = 3.7 Hz, 1H, H-1), 3.92 (t, J = 9.5 Hz, 1H, H-3), 3.82 (m, 3H,
57 Perbenzylated β-CD (2).
To a stirred solution of β-cyclodextrin (12.1 g, 10.7 mmol) in DMSO (213 mL) at 0 °C under nitrogen, NaH (60% dispersion in mineral oil, 17.9 g, 0.45 mol) was added portionwise in 15 min. Benzyl chloride (51 mL, 0.45 mol) was added dropwise in 1 h. The reaction mixture was left to proceed overnight (19 h) at ambient temperature and then neutralized by the slow addition of MeOH (55 mL) at 0 °C. Then the resulting mixture was diluted with H2O and extracted
with Et2O (3×500 mL). The combined organic layers were separated, dried
over MgSO4, and concentrated. Purification by flash column chromatography (silica gel,
pentane/Et2O = from 4/1 to 1/1) afforded the title compound as a white foam (Yield: 28.4 g, 9.4
mmol, 88%). The analytical data were in accordance with those reported previously.30 Rf = 0.21
(pentane/Et2O, 2/1, v/v).
2A-G,6A-G-tetradeca-O-benzyl-β-CD (4).
Perbenzylated β-cyclodextrin 2 (2.80 g, 0.9 mmol) and iodine (1.81g, 7.1 mmol) were suspended in DCM (396 mL, 18 mM I2) under nitrogen. After the
complete dissolution of iodine, the purple reaction mixture was cooled down to -60 °C (temperature measured inside the flask), followed by the addition of triethylsilane (1.14 mL, 7.1 mmol). The reaction mixture was allowed to warm up to -35 °C (1.5h) and was then neutralized by the addition of K2CO3. Then
the reaction mixture was washed with a saturated solution of Na2S2O3. The
water layer was extracted with DCM (2x) and the combined organic layers were washed with brine, dried over MgSO4, and concentrated. Purification using flash column chromatography
(toluene/EtOAc, 8/1 to 3/1, v/v) yielded the title compound as a white foam (Yield: 570 mg, 0.24 mmol, 27%,). TLC: Rf = 0.24 (toluene/EtOAc, 5/1, v/v). 1H NMR (400 MHz, CDCl3) δ 7.46 – 7.32 (m, 7×5H, CHarom), 7.22 – 7.12 (m, 7×5H, CHarom), 5.11 (s, 7H, 7×OH), 5.00 (d, 7H, J = 11.9 Hz, 7×CHH Bn), 4.84 – 4.73 (m, 14H, 7×H-1, 7×CHH Bn), 4.48 (d, 7H, J = 12.2 Hz, 7×CHH Bn), 4.25 (d, 7H, J = 12.2 Hz, 7×CHH Bn), 4.05 (t, 7H, J = 9.2 Hz, 7×H-3), 3.73 (m, 7H, 7×H-5), 3.63 – 3.55 (m, 7H, 7×H-6a), 3.46 (m, 21H, 7×H-2, 7×H-4, 7×H-6b). 13C-APT NMR (100 MHz, CDCl3) δ 138.1 (7×Cq), 137.6 (7×Cq), 128.9, 128.4, 128.08, 127.6, 127.5 (CHarom), 102.1 (7×C-1), 83.4 (7×C-4), 78.3 (7×C-2), 74.0 (7×CH2 Bn), 73.8 (7×C-3), 73.1 (7×CH2 Bn), 70.3 (7×C-5), 68.5 (7×C-6).
ESI-HRMS: [M-H]- calcd for C140H153O35 2395.0221 found 2395.0262.
3.5.4.3 Fucosylations of cyclodextrins
3A-O-(2,3,4-tri-O-α-L-benzylfucopyranosyl)-2A-G,6A-G-tetradeca-O-benzyl-β-CD
(Bn-Fuc-β-CD)
A mixture of donor 5 (87 mg, 0.161 mmol), Ph2SO (42
mg, 0.21 mmol) and TTBP (100 mg, 0.40 mmol) was co-evaporated 3 times with dry toluene. The residue was dissolved in DCM (0.4 mL) under nitrogen and activated molecular sieves (4Å) were added. The resulting mixture was stirred at room temperature for 20 min and then it was cooled down to -80°C (acetone cooling bath equipped with a stirring bar). Tf2O (35 µL, 0.21 mmol)
was added and the process of donor activation was monitored by TLC analysis. After 40 minutes, when TLC analysis indicated complete activation of the donor, the reaction mixture was cooled down to -85°C and a solution of acceptor 4 (48 mg, 0.020 mmol) in DCM (0.3 mL) was added in portions of 0.15 mL slowly (in 2 minutes) via the wall of the flask. Upon
58
completion of the addition, the temperature reached -80°C and was allowed to warm up to -65°C (12 min). The reaction mixture was then diluted with DCM, and neutralized by the addition of H2O and Et3N. The mixture was concentrated in vacuo and purified on size-exclusion Sephadex
LH-20 (DCM/MeOH, 1/1, v/v). The fraction of fucosylated cyclodextrins (45 mg, 70% yield) contained: 27% of octasaccharide and 37% of nonasaccharide 9, the rest contained acceptor and overfucosylated products. Rf = 0.14 (Toluene/EtOAc, 8/1, v/v). The title compound was purified
by preparative HPLC on BEH C4 column (5 mg) (RT 22.5) and purity was checked by UPLC-UV/MS (RT 14.1 min). 1H NMR (600 MHz, CDCl3) δ 7.44 – 6.96 (m, 85H, CHarom), 5.81 (d, 1H, J = 4.0 Hz, H-1Fuc), 4.96 – 4.81 (m, 10H, 9×CHH Bn, H-1Glc), 4.78 (d, 1H, J = 3.6 Hz, H-1Glc), 4.76 – 4.64 (m, 13H, 4×H-1Glc, H-5Fuc, CH2 Bn, 6×CHH Bn), 4.54 – 4.48 (m, 3H, 3×CHH Bn), 4.47 (d, J = 3.4 Hz, 1H, H-1Glc), 4.44 – 4.20 (m, 13H, CHH Bn, 5×CH2 Bn, H-3Glc, H-3Fuc), 4.18 – 4.05 (m, 4H, 3×CHH Bn, H-2Fuc), 3.98 (t, J = 9.3 Hz, 4H, 4×H-3Glc), 3.90 – 3.85 (m, 2H, H-3Glc, H-4Fuc), 3.80 (dt, 1H, J = 3.6, 7.3 Hz, H-5Glc), 3.76 – 3.71 (m, 2H, H-4Glc, H-5Glc), 3.71 – 3.67 (m, 3H, 3×H-5Glc), 3.63 – 3.49 (m, 8H, H-3Glc, H-5Glc, 3×H-6Glc), 3.49 – 3.46 (m, 1H, H-2Glc), 3.45 – 3.30 (m, 17H, 5×H-2Glc, 4×H-4Glc, 8×H-6Glc), 3.29 – 3.17 (m, 6H, H-2Glc, 2×H-4Glc, 3×H-6Glc), 1.24 (d, J = 6.5 Hz, 3H, CH3 Fuc). 13C NMR (151 MHz, CDCl3) δ 139.4, 139.2, 139.0, 139.0, 138.3, 138.1, 138.1, 138.0, 137.7, 137.6, 137.4, 136.8 (Cq); 129.1, 129.1, 128.9, 128.9, 128.8, 128.6, 128.5, 128.4, 128.4, 128.3, 128.3, 128.3, 128.2, 128.1, 128.1, 128.1, 128.0, 127.9, 127.8, 127.7, 127.7, 127.7, 127.6, 127.6, 127.5, 127.5, 127.5, 127.5, 127.4, 127.3, 127.2, 127.2, 126.9 (Carom); 102.4, 102.2, 102.0, 102.0, 101.8, 101.7 (6×C-1Glc); 99.4 (C-1Glc), 97.5 (C-1Fuc); 84.3, 83.8, 83.7, 83.3, 83.1, 83.0 (6×C-4Glc); 82.5 (C-2Glc), 79.7 (C-3Glc), 79.4 (C-4Glc), 79.0 (C-2Glc), 78.7(C-3Glc), 78.5, 78.4, 78.3, 77.9, 77.8 (5×C-2Glc), 76.2 (C-2Fuc), 75.1, 74.2, 74.2, 74.1, 74.1 (CH2 Bn); 73.9, 73.8, 73.8, 73.6 (4×C-3Glc), 73.3, 73.2, 73.2, 73.2, 73.1, 73.0 (CH2 Bn), 72.7 (C-5Glc), 72.2 (C-3Glc), 71.8 (C-3Fuc) 71.5 (CH2 Bn), 70.4, 70.3, 70.3, 70.3, 70.0, 69.9, (7×C-5Glc), 69.8, 68.9, 68.8, 68.4, 68.4, 68.3, 66.1 (7×C-6Glc), 16.5 (C-6Fuc).
ESI-HRMS: [M+2×NH4]2+ calcd for C167H190O39N2 1424.1484 found 1424.1486.
3A,3D-Di-O-(2,3,4-tri-O-α-L-benzylfucopyranosyl)-2A-G,6A-G-tetradeca-O-benzyl-β-CD (9)
A mixture of donor 5 (1.81 g, 3.35 mmol), Ph2SO
(0.881 g, 4.36 mmol), and TTBP (2.08 g, 8.39 mmol) was co-evaporated 3 times with dry toluene. The residue dissolved in dry DCM (8 mL) under nitrogen and activated molecular sieves (4Å) were added. The resulting mixture was stirred at room temperature for 20 min and then cooled down to -80 °C (acetone cooling bath equipped with a stirring bar). Tf2O (0.73
mL, 4.36 mmol) was added dropwise and the process of donor activation was monitored by TLC analysis (pentane/Et2O, 6/1, v/v, Rf of donor = 0.33). After 40
minutes when TLC analysis indicated complete activation of the donor, the reaction mixture was cooled down to -85 °C and a solution of acceptor 4 (1.0 g, 0.42 mmol, 1 eq) in dry DCM (6 mL) was added in portions of 2 mL slowly (in 10 minutes) via
the wall of the flask. Upon complete addition, the temperature reached -80 °C and the reaction mixture was allowed to warm up to -60 °C (15 min). The mixture was diluted with DCM and neutralized by the addition of H2O and Et3N. Then the crude mixture was concentrated and
purified on size-exclusion Sephadex LH-20 (DCM/MeOH, 1/1, v/v). Additional purification by column chromatography provided nonasaccharide 9 as a white foam (Yield: 898 mg, 0.28 mmol, 67%, 69% purity based on UPLC UV absorbance 208 nm) TLC: Rf = 0.19 (toluene/EtOAc, 8/1,
v/v). For the detailed analysis, the title compound was purified by preparative HPLC on BEH C4 (RT 27.1 min) and purity was checked by UPLC-UV/MS (RT 17.0 min).
59 1H NMR (600 MHz, CDCl3) δ 7.62 – 6.95 (m, 100H, CHarom), 5.97 (d, 1H, J = 4.1 Hz, H-1Fuc), 5.93 (d, 1H, J = 3.9 Hz, H-1Fuc), 5.09 – 4.72 (m, 29H, 8×CH2 Bn, 5×H-1Glc, 2×H-5Fuc), 4.71 – 4.50 (m, 16H, 2×H-1Glc, 7×CH2 Bn), 4.50 – 4.12 (m, 38H, 5×CH2 Bn, 2×H-3Glc, 2×H-2Fuc, 2×H-3Fuc), 4.11 – 3.95 (m, 6H, 4×H-3Glc, 2×H-4Fuc), 3.94 – 3.83 (m, 4H, 2×H-4Glc, 2×H-5Glc), 3.79 – 3.63 (m, 7H, H-3Glc, 5×H-5Glc), 3.63 – 3.22 (m, 26H, 7×H-2Glc, 5×H-4Glc, 14×H-6Glc), 1.38 (d, 3H, J = 6.4 Hz, H-6Fuc), 1.34 (d, 3H, J = 6.5 Hz, H-6Fuc). 13C NMR (150 MHz, CDCl3) δ 139.4, 139.2, 139.2, 139.1, 139.1, 138.9, 138.3, 138.3, 138.2, 138.1, 138.1, 137.9, 137.9, 137.7, 137.6, 137.5, 137.4, 137.3, 136.8, 136.8, (Cq), 128.5, 128.4, 128.4, 128.4, 128.4, 128.3, 128.3, 128.2, 128.2, 128.2, 128.1, 128.1, 128.1, 127.8, 127.8, 127.7, 127.7, 127.7, 127.7, 127.6, 127.5, 127.5 (Carom), 102.6, 102.2, 102.1, 101.9, 101.6 , 99.5, 99.4 (7×C-1Glc), 97.5, 97.3 (2×C-1Fuc), 84.5, 84.1, 83.9, 83.8, 83.6, 83.0, 82.5 (2×C-2Glc, 5×C-4Glc), 79.8, 79.7, 79.4, 79.3, 79.1, 79.1, 78.8, 78.7, 78.6, 78.3, 78.1, 78.0, 77.4 (5×C-2Glc, 3×C-3Glc, 2×C-4Glc), 76.5, 76.1 (2×C-2Fuc), 75.1, 75.0, 74.3, 74.2, 74.2, 74.1, 74.1, 74.0, 73.9, 73.3, 73.1, 73.1, 73.1, 73.0, 72.9, 72.9, 72.8, 72.6, 72.5, 72.1, 72.1, 71.7, 71.4, 71.4 (5×C-3Glc, 20×CH2 Bn); 70.4, 70.3, 70.1, 70.0, 69.9, 69.8(7×C-5Glc), 69.4, 69.0, 69.0, 68.5, 68.4, 68.2, 68.1 (7×C-6Glc); 66.2, 66.2 (2×C-5Fuc), 16.7, 16.6 (2×C-6Fuc).
ESI-HRMS: [M+NH4]+ calcd for C194H214O43N1 3247.4651 found 3227.4560.
3A-O-(α-L-fucopyranosyl)-β-CD (Fuc1-β-CD)
Octasaccharide BnFuc1-β-CD, as isolated from
preparative HPLC, was dissolved in a THF/H2O mixture
(2.5 ml/0.2 mL) and excess of Pd/C was added. The mixture was purged with nitrogen, followed by purging with hydrogen, and the mixture was left to react overnight under a blanket of hydrogen gas. The mixture was filtered over a pad of celite, concentrated, and lyophilized (1 mg). 1H NMR (600 MHz, D2O) δ 5.54 (d, 1H, J = 3.8 Hz, H-1Fuc), 5.16 (d, 1H, J = 3.8 Hz, H-1Glc), 5.12 – 5.04 (m, 6H, 6×H-1Glc), 4.46 (q, 1H, J = 6.7 Hz, H-5Fuc), 4.21 – 4.15 (m, 1H, H-6Glc), 4.15 – 4.10 (m, 1H, H-3Glc), 4.00 – 3.93 (m, 8H, 6×H-3Glc, H-6Glc, H-4Fuc), 3.92 – 3.80 (m, 22H, H-4Glc, 7×H-5Glc, 12×H-6Glc, H-2Fuc, H-3Fuc), 3.71 – 3.55 (m, 13H, 7×H-2Glc, 6×H-4Glc ), 1.26 (d, 3H, J = 6.5 Hz, H-6Fuc). 13C NMR (150 MHz, D2O) δ 102.0, 101.9, 101.8, 101.7, 101.6 (6×C-1Glc); 99.9 (C-1Glc), 98.8 (C-1Fuc), 81.1, 81.0, 80.9, 80.8 (6×C-4Glc), 78.7 (C-4Glc), 77.3 (C-3Glc); 73.2, 73.1, 73.0, 73.0, (6×C-3Glc), 72.6, 72.1, 71.9, 71.9, 71.8, 71.7, 71.6, 71.6, 71.4, 71.2 (7×C-2Glc, 7×C-5Glc, C-3Fuc), 69.2
(C-4Fuc), 68.9 (C-2Fuc), 67.8 (C-5Fuc), 60.5, 60.2, 60.1 (7×C-6Glc); 15.3 (C-6Fuc).
60
3A,3D-Di-O-(α-L-fucopyranosyl)-β-CD (10)
Nonasaccharide 9 (830 mg, 0.26 mmol) was dissolved in THF (10 mL) and H2O (1 mL) and Pd/C (6 g) was
added. The reaction mixture was purged with nitrogen followed by purging with hydrogen. The reaction was left to stir over 3 days under a blanket of hydrogen gas, after which the mixture was filtered over celite, concentrated, and lyophilized to yield the title compound as a white solid (Yield: 275 mg, 0.19 mmol, 75%, 69% purity). RT on Cortecs HILIC 3.5 min.
1H NMR (600 MHz, D2O) δ 5.56 (d, 1H, J = 3.8 Hz, H-1Fuc), 5.54 (d, 1H, J = 3.9 Hz, H-1Fuc), 5.19 (d, 1H, J = 4.1 Hz, H-1Glc), 5.18 (d, 1H, J = 3.9 Hz, H-1Glc), 5.15 (app t, 2H, J = 3.5 Hz, 2×H-1Glc), 5.12 (d, 1H, J = 3.7 Hz, H-1Glc), 5.08 (app t, 2H, J = 3.1 Hz, 2×H-1Glc), 4.47 (q, J = 6.2, 5.5 Hz, 2H, 2×H-5Fuc), 4.21 (dt, 2H, J = 4.1, 12.6 Hz, 2×H-6Glc), 4.16 (t, 2H, J = 8.3 Hz, 2×H-3Glc), 4.06 – 3.98 (m, 6H, 3×H-3Glc, 2×H-6Glc, 2×H-4Fuc), 3.96 – 3.80 (m, 35H, 2×H-3Glc, 2×H-4Glc, 7×H-5Glc, 10×H-6Glc, 2×H-2Fuc, 2×H-3Fuc), 3.75 – 3.67 (m, 5H, 5×H-2Glc), 3.65 – 3.54 (m, 7H, 2×H-2Glc, 5×H-4Glc), 1.28 (d, 6H, J = 6.6 Hz, 2×H-6Fuc). 13C NMR (150 MHz, D2O) δ 101.9, 101.8, 101.7, 101.5, 101.2 (5×C-1Glc), 99.5, 99.3 (2×C-1Glc), 99.0, 98.9 (2×C-1Fuc), 81.3, 81.1, 81.0, 80.4, (5×C-4Glc), 78.5, 78.4 (2×C-4Glc), 77.5, 77.2 (2×C-3Glc); 73.2, 73.2, 73.1, 72.8, 72.6 (5×C-3Glc), 72.1, 72.0, 72.0, 71.9, 71.7, 71.6, 71.4, 71.3, 71.2
(7×C-5Glc, 7×C-2Glc, 2×C-3Fuc), 69.3 (2×C-4Fuc), 69.0, 68.9 (2×C-2Fuc), 67.9, 67.8 (2×C-5Fuc), 60.7, 60.4,
60.3, 60.2, 60.2 (7×C-6Glc), 15.3 (2×C-6Fuc).
ESI-HRMS: [M-H]- calcd for C54H89O43 1425.4772 found 1425.4810.
3.6
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